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. 2023 Jul;299(7):104906.
doi: 10.1016/j.jbc.2023.104906. Epub 2023 Jun 9.

Identification of a novel mitochondria-localized LKB1 variant required for the regulation of the oxidative stress response

Ivan Tan  1 , Shengli Xu  2 , Jianxin Huo  1 , Yuhan Huang  1 , Hong-Hwa Lim  3 , Kong-Peng Lam  4
Affiliations

Identification of a novel mitochondria-localized LKB1 variant required for the regulation of the oxidative stress response

Ivan Tan et al. J Biol Chem. 2023 Jul.

Abstract

The tumor suppressor Liver Kinase B1 (LKB1) is a multifunctional serine/threonine protein kinase that regulates cell metabolism, polarity, and growth and is associated with Peutz-Jeghers Syndrome and cancer predisposition. The LKB1 gene comprises 10 exons and 9 introns. Three spliced LKB1 variants have been documented, and they reside mainly in the cytoplasm, although two possess a nuclear-localization sequence (NLS) and are able to shuttle into the nucleus. Here, we report the identification of a fourth and novel LKB1 isoform that is, interestingly, targeted to the mitochondria. We show that this mitochondria-localized LKB1 (mLKB1) is generated from alternative splicing in the 5' region of the transcript and translated from an alternative initiation codon encoded by a previously unknown exon 1b (131 bp) hidden within the long intron 1 of LKB1 gene. We found by replacing the N-terminal NLS of the canonical LKB1 isoform, the N-terminus of the alternatively spliced mLKB1 variant encodes a mitochondrial transit peptide that allows it to localize to the mitochondria. We further demonstrate that mLKB1 colocalizes histologically with mitochondria-resident ATP Synthase and NAD-dependent deacetylase sirtuin-3, mitochondrial (SIRT3) and that its expression is rapidly and transiently upregulated by oxidative stress. We conclude that this novel LKB1 isoform, mLKB1, plays a critical role in regulating mitochondrial metabolic activity and oxidative stress response.

Keywords: DNA damage; cellular localization; exon; intron; isoforms; mitochondrial respiration.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflict of interest with the contents of this article.

Figures

Figure 1
Figure 1
Identification of a novel exon with an alternative initiation codon embedded in intron one of LKB1 gene.A, schematic diagram of LKB1 gene depicting the organization of various exons and introns. The original exon one is renamed 1a and the novel exon (in green) described in this study is named 1b. Genomic sequence of the putative 131 bp novel exon 1b flanked by short intronic sequences. The putative exon sequence (green, upper case letters), ATG start codon (red, upper case letters), intronic sequences (black, lower case letters), and the conserved 5′-splice donor and 3′-splice acceptor sites (gt-ag, red lower-case letters) are shown. B, BM819015 EST obtained from the NCBI database with sequences corresponding to LKB1 exon 1a (blue), exon 2 (orange), and exon 1b (green). The canonical ATG start codon encoded by exon 1a is in red and underlined and the alternative ATG start codon in exon 1b is in red. The premature stop codon encoded in exon 1b is boxed. Conserved Gs at −3 and +4 positions of the Kozak motif of the alternative initiation site are bolded and underlined. C, organization of the spliced transcripts of the canonical LKB1 and the novel variant with exon 1b (green) insertion (sequence from BM819015). The amino acid sequence translated from the canonical ATG start site is shown. Note that translation is terminated prematurely by the stop codon encoded in exon 1b. The amino acid residue N (black) resulted from the joining of exons 1a and 1b. D, organization of the sequence from BM819015 EST and the amino acid sequence translated from the alternative start codon in exon 1b. The amino acid residue W (black) resulted from the joining of exons 1b and 2. Comparison of the translation initiation site (TIS) in exon 1b and the conserved Kozak motif. E, verifications of the spliced transcripts corresponding to the canonical and novel LKB1 variants. Relative annealing positions of primers used for RT-PCR are shown. RT-PCR products amplified from cDNA synthesized from U2OS cells, using primer pairs F1/R1, F2/R1, and F1/R2, for the detection of the transcript of the novel LKB1 variant with exon 1b insertion. The expected sizes of the PCR products derived from the various primers are shown. A non-specific PCR product is marked by ∗.
Figure 2
Figure 2
Generation of a novel LKB1 isoform from alternative translation start site encoded in exon 1b.A, schematic diagram showing the genomic and protein domain structures of human LKB1. Corresponding exon and protein subdomains are connected by dotted lines. Translation initiation from the use of the canonical ATG start codon encoded in exon 1a produces the full-length LKB1L protein (433 amino acids). Translation initiation from the alternative ATG start codon in exon 1b produces the novel LKB1 variant (359 amino acids). The amino acid sequence encoded by exon 1b is shown. B, Western blot analysis of LKB1 protein. Triton X-100-soluble (20 mg) and -insoluble (50 mg) fractions of U2OS cells were resolved on SDS-PAGE and probed with two different LKB1 antibodies as indicated. The anti-tubulin-a and anti-IDH2 blots were included as controls for the fractionation of cellular lysates. C, in vitro kinase assay of overexpressed novel LKB1 variant. Full-length (WT) and kinase domain (CAT) of novel LKB1 variant were immunoprecipitated with FLAG antibody-coated agarose and incubated with Histone H1 as substrate. Proteins were resolved on SDS-PAGE and the phosphorylation of histone H1 was examined using an anti-phospho-Thr antibody. aa, amino acid; C-ter, C-terminal; NLS, nuclear localization signal.
Figure 3
Figure 3
Mitochondria targeting of novel LKB1 variant.A, prediction of a mitochondrial targeting sequence in the N-terminus of novel LKB1 variant using online MITOPROT and TargetP 1.1 analysis software. The sequence corresponding to N-terminal 60 amino acid residues of the novel LKB1 variant was used in the analyses. Amino acids encoded by exon 1a (green), exon 1b (orange) and residue resulting from merging of exons 1a and b (black) are shown. B, enrichment of the smaller novel LKB1 variant in the mitochondrial fraction. Cytosolic and mitochondria-enriched fractions of U2OS cells were resolved on SDS-PAGE and probed with an anti-LKB1 antibody. The anti-VDAC and anti-tubulin-α blots were included as controls for the fractionation of cell lysates. C, colocalization of novel LKB1 variant with mitochondria-resident ATP Synthase. U2OS cells were permeabilized with 0.1% Triton X-100 for 10 to 15 s to deplete cytosolic LKB1FL, fixed with cold methanol at −20 °C and immune-stained with anti-LKB1 and anti-ATP Synthase antibodies overnight. Line scan analysis of boxed cell is shown. Scale bar: 10 μm. D, localization of over-expressed LKB1 variant to mitochondria. Cos-7 cells were co-transfected with HA-tagged SIRT3 and FLAG-tagged mLKB1 variant or FLAG-tagged LKB1L or FLAG-tagged N-terminal putative mitochondria transit peptide. Thereafter, cells were stained with DAPI (blue) and fluorochrome-conjugated anti-HA (red) and anti-FLAG (green) antibodies to visualize the overexpressed proteins and peptides. Scale bars: 10 μm. Images were acquired using Olympus Fluoview1000 confocal microscope.
Figure 4
Figure 4
Expression of mLKB1 is regulated by oxidative stress.A, upregulation of mLKB1 protein expression by H2O2 treatment. U2OS cells were treated with 1 mM H2O2 for 1 and 2 h, fractionated into Triton X-100-soluble and -insoluble fractions, and probed with the two separate LKB1 antibodies as indicated. B, increased mLKB1 in mitochondria upon H2O2 stimulation. U2OS cells were treated with 1 mM H2O2 for 1 h, then permeabilized by Triton-X 100-containg PEM buffer and immunostained with antibodies against LKB1 and ATP Synthase overnight. Images were acquired using Olympus Fluoview1000 confocal microscope. Scale bars: 10 μm. C, examination of mLKB1 transcripts in H2O2 treated U2OS cells. Total RNAs obtained from U2OS cells treated with H2O2 for 1 and 2 h were subjected to RT-PCR using the F1/R1 primer pair in Figure 1E to amplify LKB1L and mLKB1 transcripts.
Figure 5
Figure 5
mLKB1 is required for the regulation of mitochondrial metabolic activity.A, A549 cells possess only mLKB1 isoform. Triton X-100-soluble and -insoluble fractions of U2OS and A549 cells were resolved on SDS-PAGE and probed with an anti-LKB1 antibody. The anti-tubulin-a or anti-IDH2 blots were included as controls for the fractionation of cell lysates. Protein extracts loaded were 20 mg for lanes one and 3 and 50 mg for lanes two and 4. B, detection of mLKB1 in a mitochondria-enriched fraction of A549 cells. Various subcellular fractions obtained from A549 cells were resolved on SDS-PAGE and probed with anti-LKB1 antibody and other various antibodies against specific organelle to mark subcellular fractions: anti-EGFR for membrane; anti-PARP for nucleus; anti-Tub-a for cytosol and anti-VDAC for the mitochondrial fraction. Cyto I, 12 kg supernatant; Cyo II, 450 kg supernatant. C, Western blot analysis of mLKB1 knockdown in A549 cells. Cells were transfected with scrambled siRNA as control (si-Ctrl) or mLKB1-specific siRNA (si-mLKB1). Triton X-100-insoluble fractions were prepared and probed for mLKB1 expression with an anti-LKB1 antibody. The anti-IDH2 blot was included as a control. D, real-time analysis of the Oxygen Consumption Rate (OCR) of A549 cells transfected with scrambled siRNA (si-Ctrl, blue), mLKB1-specific siRNA (si-mLKB1, red), or mLKB1 expression vector (mLKB1 OE, green). OCR was measured with the consecutive addition of oligomycin (1 mM), mitochondrial uncoupler FCCP (500 nM), and inhibitors of the mitochondrial electron-transport complex I and III, rotenone and antimycin (R&A, 500 nM each). E, quantifications of basal mitochondrial respiration, ATP production, spare respiratory capacity, and proton leak. Data are represented as Mean ± SD (n = 6), and samples were compared using independent Student's t-tests; ∗∗p < 0.01; ∗∗∗p < 0.001.
Figure 6
Figure 6
mLKB1 regulates mitochondrial oxidative stress.A, increased oxidative stress in mLKB1-knockdown A549 cells. Visualization (left panel) and quantification (right panel) of mitochondrial oxidative stress in control and mLKB1 knockdown cells using MitoSOX staining. Scale bar: 50 μm. B, enhanced DNA damage in mLKB1-knockdown A549 cells. Western blot analysis of the levels of H2O2-induced DNA damage in A549 cells transfected with si-Ctrl or si-mLKB1, co-transfected (24 h later) with either an empty pBabe vector, FLAG-LKB1L/HA-STRADα constructs or an untagged siRNA-resistant mLKB1 construct (rescue) as indicated, using anti-pH2AX Ser139 antibody. The expression of endogenous mLKB1, reconstituted mLKB1, and LKB1L were detected by an anti-LKB1 antibody. The anti-Histone H3 blot was included as a loading control. C, quantification and statistical analysis of data in (B). Data are represented as Mean ± SD (n = 3), and samples were compared using independent Student's t-tests; ∗∗∗p < 0.001; ∗∗p < 0.01. D, visualization of DNA damage in A549 cells depleted of, or over-expressing mLKB1 in response to H2O2. A549 cells transfected with si-Ctrl or si-mLKB1 were co-transfected with untagged siRNA-resistant mLKB1 construct, followed by 0.5 mM H2O2 treatment for 1 h and stained with DAPI, anti-LKB1, and anti-pH2AX Ser139 antibodies. Arrows indicate transfected cells overexpressing mLKB1. Scale bars: 25 μm. Images were acquired using Olympus Fluoview1000 confocal microscope. E, enhanced DNA damage in mLKB1-knockdown U2OS cells. Western blot analysis of the levels of H2O2-induced DNA damage in U2OS cells transfected with si-Ctrl or si-mLKB1 using anti-pH2AX Ser139 antibody. Triton X-100 soluble and insoluble fractions were analyzed by separate antibodies as indicated. F, quantification and statistical analysis of data in (E). Data are represented as Mean ± SD (n = 3), and samples were compared using independent Student's t tests; ∗p < 0.05; ∗∗p < 0.01.
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